The role of glucocerebrosidase and chaperone_mediated autophagy in Parkinsons dis

描述（由申请人提供）：多巴胺能神经元中的突触核蛋白聚集是帕金森病（PD）病理的标志。PD最常见的遗传危险因素是葡萄糖脑苷酶（GBA）突变，多达7%的PD患者携带该突变。迄今为止的工作主要集中在GBA酶活性的丧失上，这可能导致-突触核蛋白的积累和/或聚集。然而，有证据表明，不正确折叠的突变体GBA也可能导致独立于酶活性丧失的¿-突触核蛋白聚集。我们的中心假设是突变型GBA从溶酶体错靶到细胞质，这干扰了伴侣介导的自噬（CMA）对¿-突触核蛋白的降解。我们将结合生物化学、细胞生物学、小鼠遗传学和神经病理学技术，在几个系统中检验这一假设，包括分离的溶酶体、小鼠模型、死后的人类大脑和患者皮肤活检的成纤维细胞。我们的研究策略分为三个具体目标：目标1将通过使用分离的溶酶体、纯化的突变体GBA和-synuclein蛋白在体外系统中检测CMA的每个步骤，研究突变体GBA减弱CMA并导致-synuclein积累的机制。Aim 2将通过在神经元培养中使用一种新的CMA报告基因，研究CMA是否会在GBA突变小鼠模型中发生改变，并确定GBA突变小鼠模型和死后PD脑中CMA机械成分的蛋白质水平是否会发生变化。Aim 3将使用报告基因测定来自GBA突变PD患者、无GBA突变PD患者和年龄匹配对照的皮肤成纤维细胞中的CMA活性。这些数据将为突变型GBA是否导致CMA功能障碍导致¿-synuclein聚集，以及CMA功能障碍是否是PD的典型特征提供证据。该研究将有助于未来确定基于机制的生物标志物和治疗靶点。这份K08提案还概述了一个详细的5年培训计划，包括具体的正式课程和对候选人的结构化指导。建议的工作将在哥伦比亚大学神经内科进行，这是一个培养医生科学家的良好环境。他将在博士的指导下接受必要的培训。David Sulzer， Ana Maria Cuervo和Karen Marder， PD领域的世界知名研究者，并获得了成为独立研究者所需的技能。候选人的长期目标是成为一名转化医师兼科学家，研究帕金森病的病理机制，并开发临床适用的帕金森病生物标志物。

DESCRIPTION (provided by applicant): Synuclein aggregation in dopaminergic neurons is a hallmark of Parkinson's disease (PD) pathology. The most common genetic risk factor for PD is glucocerebrosidase (GBA) mutation with as many as 7% of PD patients carrying this mutation. Work to date has primarily focused on the loss of GBA enzymatic activity that could contribute to ¿-synuclein accumulation and/or aggregation. However, evidence has indicated that improperly folded mutant GBA could also contribute to the ¿-synuclein aggregation independent of the loss of enzymatic activity. Our central hypothesis is that mutant GBA is mistargeted from lysosomes to the cytosol, which interferes with ¿-synuclein degradation by chaperone-mediated autophagy (CMA). We will test this hypothesis with a combination of biochemical, cell biological, mouse genetics, and neuropath logical techniques in several systems including isolated lysosomes, mouse models, post-mortem human brain, and fibroblasts from patients' skin biopsy. Our research strategies are divided into three specific aims: Aim 1 will investigate the mechanism by which mutant GBA attenuates CMA and leads to ¿-synuclein accumulation by examining each step of CMA in an in vitro system using isolated lysosomes, purified mutant GBA and ¿- synuclein proteins. Aim 2 will study whether CMA alteration occurs in GBA-mutant mouse models by using a novel CMA reporter in neuronal cultures and determine whether the protein levels of CMA machinery components change in GBA-mutant mouse models and post-mortem PD brain with GBA mutations. Aim 3 will determine CMA activity using the reporter in the skin fibroblasts from PD patients with GBA mutations, PD patients without GBA mutations, and age-matched controls. These data will provide evidence on whether mutant GBA causes CMA dysfunction, leading to ¿-synuclein aggregation, and whether CMA dysfunction is a typical feature of PD. The proposed study could contribute to the future identification of mechanism-based biomarker and therapeutic targets. This K08 proposal also outlines a detailed 5-year training program with specific formal coursework and structured mentoring for the candidate, Sheng-Han Kuo, M.D. The proposed work will be carried out in the Department of Neurology at Columbia University, an excellent environment for training physician-scientists. He will receive the necessary training under the mentorship of Drs. David Sulzer, Ana Maria Cuervo, and Karen Marder, world-renowned investigators in the PD field and acquire necessary skill set to become an independent researcher. The long-term goal of the candidate is to be a translational physician-scientist to investigate the mechanism underlying PD pathology and to develop clinical applicable biomarkers for PD.